Compositions And Methods For Immunodominant Antigens

ABSTRACT

Contemplated compositions, devices, and methods comprise immunodominant antigens from selected human pathogens ( Burkholderia pseudomallei, Borrelia burgdorferi, Brucella melitensis, Chlamydia muridarum, Coxiella burnetii, Francisella tularensis , human Herpes virus 1 and 2,  Mycobacterium tuberculosis, Plasmodium falciparum , and Vaccinia virus) can be used as a vaccine, as diagnostic markers, and as therapeutic agents. In particularly preferred aspects, the antigens have quantified and known relative reactivities with respect to sera of a population infected with the pathogen, and have a known association with a disease parameter.

This application is a divisional application of U.S. application Ser.No. 12/447,620, filed Dec. 3, 2009, which is a U.S. National Phasefiling of International Application No. PCT/US07/23299, filed Nov. 1,2007, which claims priority to U.S. Provisional Application No.60/856,217, filed Nov. 1, 2006.

FIELD OF THE INVENTION

The field of the invention is immunodominant antigens from pathogens,especially as they relate to their use in diagnostic and therapeuticcompositions and methods.

BACKGROUND OF THE INVENTION

Antigens for vaccination and/or diagnostic purposes are typically singleantigens from a pathogen, or complex mixtures of multiple and unknownantigens of a pathogen such as inactivated bacteria or viruses.Depending on the particular type of pathogen, single antigens mayprovide a quantifiable signal in diagnostic test. However, due tovariations among individuals in their immune response profiles, singleantigen tests are often not sufficient to obtain useful diagnosticinformation with useful specificity and sensitivity. In addition, wherethe single antigen is used as a vaccine, variability of individualimmune response and potential prior exposure often limit usefulness ofsingle antigens. Finally, while some complex mixtures of multiple andunknown antigens are useful for vaccine development, they typicallycarry the risk of adverse reactions, or even reactivation of thepathogen.

More recently, multivalent vaccine preparations have become availablewhere in a single dose, multiple and distinct antigens, from multipleand distinct serotypes, of a single pathogenic organisms were combined(Prevnar™: Heptavalent vaccine against Streptococcus pneumoniae capsularserotypes 4, 6B, 9V, 14, 18C, 19F, and 23F). While such mixedpreparations tend to provide a broader range of protection againstdifferent serotypes, various difficulties nevertheless remain. Mostsignificantly, where a single antigen fails to elicit an immuneresponse, coverage to the corresponding serotype is not present. Thus,combination of single defined antigens from several serotypes merelycombines benefits and problems associated with the single antigens.Moreover, none of the heretofore known antigens is generally applicableto differentiate among stages, secondary infections, etc., as the signalis either impossible to deconvolute (e.g., compound signal frominactivated pathogen) or only provides a single data point.

Therefore, while numerous methods of identification and use of antigensare known in the art, all or almost all of them suffer from one or moredisadvantages. Consequently, there remains a large, unmet need toprovide improved compositions and methods of antigens for diagnostic andtherapy.

SUMMARY OF THE INVENTION

The present invention is directed to immunodominant antigens fromvarious human pathogens wherein the antigens have predeterminedreactivities to serum of a population of patients infected with thepathogen. Thus, immunodominant antigens will have a statistically highprobability to elicit an immune response in a relatively large group ofpatients. Further, where the antigens are determined from selectedsub-populations (e.g., primary infection, secondary infection,recovering, chronic etc.), the antigens also have a known associationwith a disease parameter.

In one aspect of the inventive subject matter, an antigen compositioncomprises two or more immunodominant antigens of a pathogenic organismand are associated with a carrier, wherein the antigens have quantifiedand known relative reactivities with respect to sera of a populationinfected with the organism, and wherein the antigens have a knownassociation with a disease parameter. Most preferably, the antigens arepolypeptides and are encoded by nucleic acids having a sequenceaccording to SEQ ID NO:1 to SEQ ID NO:1150 (or comprise fragmentsthereof).

Thus, in some aspects, the pathogenic organism is Burkholderiapseudomallei, and the antigens are encoded by nucleic acids having asequence according to SEQ ID NO:966 to SEQ ID NO:1150, or the pathogenicorganism is Borrelia burgdorferi, and the antigens are encoded bynucleic acids having a sequence according to SEQ ID NO:546 to SEQ IDNO:637. In further aspects, the pathogenic organism is Chlamydiamuridarum, and the antigens are encoded by nucleic acids having asequence according to SEQ ID NO:1 to SEQ ID NO:134, or the pathogenicorganism is Coxiella burneti, and the antigens are encoded by nucleicacids having a sequence according to SEQ ID NO:678 to SEQ ID NO:713. Inyet further aspects, the pathogenic organism is Francisella tulare, andthe antigens are encoded by nucleic acids having a sequence according toSEQ ID NO:663 to SEQ ID NO:677, or the pathogenic organism is humanHerpes virus 1, and the antigens are encoded by nucleic acids having asequence according to SEQ ID NO:638 to SEQ ID NO:654, or the pathogenicorganism is human Herpes virus 2, and the antigens are encoded bynucleic acids having a sequence according to SEQ ID NO:655 to SEQ IDNO:662. In still further aspects, the pathogenic organism isMycobacterium tuberculosis, and the antigens are encoded by nucleicacids having a sequence according to SEQ ID NO:749 to SEQ ID NO:965, orthe pathogenic organism is Plasmodium falciparum, and the antigens areencoded by nucleic acids having a sequence according to SEQ ID NO:135 toSEQ ID NO:445. In yet further aspects, the pathogenic organism isBrucella melitensis, and the antigens are encoded by nucleic acidshaving a sequence according to SEQ ID NO:446 to SEQ ID NO:545, or thepathogenic organism is Vaccinia virus, and the antigens are encoded bynucleic acids having a sequence according to SEQ ID NO:714 to SEQ IDNO:748.

It is further contemplated that the known reactivities may becharacterized by a variety of factors, however, it is particularlypreferred that the known reactivities are characterized by strength ofimmunogenicity and/or time course of the infection. Similarly, theparticular parameter may vary among different pathogens, however, it isgenerally preferred that the parameter is a previous exposure to thepathogen, the duration of exposure to the pathogen, a chronic infection,at least partial immunity to infection with the pathogen, and/or anexpected positive outcome upon treatment.

In another aspect of the inventive subject matter, the carrier is apharmaceutically acceptable carrier, and the composition is formulatedas a vaccine. In such aspects, it is generally preferred that thevaccine comprises multiple (e.g., at least two, four, or six) antigens,which may be from the same pathogen or distinct (serotypical or species)pathogens. Depending on the particular pathogen, it is contemplated thatthe antigens or fragments thereof are at least partially purified and/orrecombinant.

Alternatively, the carrier may also be a solid carrier, and theplurality of antigens is disposed on the carrier in an array. In sucharrays, it is generally preferred that the antigens are from at leasttwo distinct pathogens, and/or have at least two distinct knownreactivities and/or parameters. It is further contemplated that theantigens or fragments thereof may be in crude expression extracts, inpartially purified form (e.g., purity of less than 60%), or in highlypurified form (purity of at least 95%). The antigens in such arrays maybe recombinant or native. Alternatively, solid phases need not belimited to planar arrays, but may also include beads, columns,dipstick-type formats, etc.

Various objects, features, aspects and advantages of the presentinvention will become more apparent from the following detaileddescription of preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an exemplary matrix depicting antibody signals for selectedantibody types with respect to potential antigens of an exemplarypathogen.

FIG. 2 is a graph illustrating the relative scarcity of antigenrecognition of selected antibody types.

FIGS. 3A and 3B are exemplary graphs depicting the average signalintensities of a plurality of immune responses against various antigensof primary (A) and secondary (B) infections.

FIGS. 4A and 4B are exemplary scans of diagnostic arrays comprisingmultiple immunodominant antigens of multiple distinct pathogensdepicting approximate quantities of antigens deposited (A) and theirreaction against human serum of a patient infected with a pathogen.

DETAILED DESCRIPTION

The inventors have discovered numerous immunodominant antigens from avariety of human pathogens capable of casing infection in humans,including: Burkholderia pseudomallei, Borrelia burgdorferi, Brucellamelitensis, Chlamydia muridarum, Coxiella burnetii, Francisellatularensis, human Herpes virus 1 and 2, Mycobacterium tuberculosis,Plasmodium falciparum, and Vaccinia virus. Immunodominant antigensaccording to the inventive subject matter are encoded by nucleic acidshaving a sequence according to SEQ ID NO:1 to SEQ ID NO:1150, and it isgenerally contemplated that such antigens can be used by themselves, ormore preferably, in combination with other antigens (typically alsoimmunodominant antigens) in the manufacture of a diagnostic devices,therapeutic compositions, and vaccines.

As used herein, the term “immunodominant antigen” refers to an antigenthat elicits in at least one stage of the disease production of one ormore types of antibodies (e.g., IgG, IgA, IgE, IgM, etc.) in at least40%, more typically at least 70%, and most typically at least 90% of apopulation exposed to the antigen, and wherein, when compared to otherantigens of the same pathogen, the average binding affinity and/oraverage quantity of the antibodies produced in the patient in at leastone stage of the disease is at least in the upper tertile, moretypically upper quartile, and most typically upper quintile. Mosttypically, the average binding affinity and/or average quantity of theantibodies is reflected in the signal intensity and signal intensity cantherefore be used as a surrogate marker for average binding affinityand/or average quantity of the antibodies. In further aspects, preferredimmunodominant antigens are also characterized by a response in the testgroup that is considered statistically significant when compared withcontrol signal intensity, wherein the significance level p is preferablyequal or less than 0.1, more preferably equal or less than 0.05, andmost preferably equal or less than 0.01.

In one aspect of the inventive subject matter, immunodominant antigensare identified from a proteome screen against sera of a population thathas been previously exposed to the pathogen. Most preferably, thepopulation is subdivided in several sub-populations to reflect variousdisease parameters, which can then be correlated with the so identifiedantigens. It is still further preferred that the screening also providesdata on relative reactivities with respect to the antigens and sera ofthe populations/sub-populations.

With respect to the above pathogens, it is generally preferred that atleast part of the pathogen's genome is obtained and all potential openreading frames and splice mutations thereof are determined in silico.Once the potential genes are identified, suitable primers are determinedto provide amplicons of the entire Open Reading Frames (ORFs), or, lesspreferably, portions thereof, wherein the primers are preferablydesigned to allow facile subcloning into an expression system. Mostpreferably, the subcloning uses recombinase-based subcloning usingunpurified PCR mixtures to avoid cloning bias, and the so obtainedrecombinant plasmids are polyclonally multiplied, which enables unbiasedpresentation of the amplicons. It is still further particularlypreferred that the plasmid preparations are then subjected to an invitro transcription/translation reaction to thereby provide therecombinant ORF peptide, which is then spotted or otherwise immobilizedonto a suitable addressable carrier (e.g., membrane, bead, etc.).

It should be recognized that the so prepared proteomes can then beexposed to serum of a population of control individuals and/orpopulation of individuals that are known to have current or previousexposure to the above pathogen from which the ORFs were prepared.Antibodies of the serum that bind to one or more of the ORFs are thendetected using well known methods (e.g., secondary antibodies). In thismanner, the entire proteome of the pathogen can be rapidly assessed forimmunogenicity and potential binding with antibodies in serum. Variouspreferred aspects, compositions, and methods of proteome preparation aredisclosed in our International patent application with the publicationnumber WO 06/088492, which is incorporated by reference herein.

Therefore, and among various other advantages, it should be especiallyrecognized that contemplated compositions and methods presented hereinwill allow for preparation of vaccines and diagnostic compositionscomprising a plurality of antigens with known and predetermined affinityto target ORFs of a pathogen. As individual immune systems are known toexhibit significant variation with respect to antigen recognition,methods and compositions contemplated herein will allow statisticallysupported antigen identification to identify immunodominant antigens ina population of patient. Consequently, multiple targets can be used toelicit an immune response and/or detect a prior exposure, even where oneor more of the targets may be evasive for detection or provide only aweak response.

With respect to the immunodominant sequences identified herein, itshould be further appreciated that the sequences need not be completeORFs, but that suitable sequences may also be partial sequences (e.g.,synthetic, recombinant or isolated) that typically comprise at leastpart of an antigenic epitope. Thus, sequences contemplated herein may beidentified as DNA sequences encoding the antigenic peptide (partial orentire ORF), or may be identified as peptide sequence (or homologsthereof). Similarly, chemically modified antigens, and/or orthologs ofthe polypeptides presented herein are also deemed suitable for useherein.

It should be particularly noted that while proteome screening willprovide a plurality of antigens as potentially useful molecules fordiagnosis, vaccination, and/or therapy, such an approach only provides araw cut of (a plurality) of individual responses. Thus, as mostindividual immune reactions towards the same pathogen elicit asignificantly distinct profile of antibodies (e.g., depending on diseasestage, previous exposure, and/or inter-individual variability), resultsobtained from such screening are typically inhomogeneous. Consequently,variability of the individual immune responses and variability of thequantity of recombinant protein in the array must be taken intoconsideration to obtain meaningful results.

Therefore, it should be appreciated that filtering of raw data willresult in a collection of antigens with quantified and known relativereactivities with respect to sera of a population infected with thepathogen. Moreover, it should be noted that as signals may be specificto a particular stage in the course of an infection, relativereactivities may be indicative of the time course of the infection,and/or relative reactivities may represent differences in the strengthof immunogenicity of the particular antigen (or quantity of depositedantigen in the screening assay). Additionally, it should be particularlyrecognized that depending on the choice of the specific patientpopulation, the tested sera will reflect the immune status of apopulation that is characterized by one or more parameters of thedisease. For example, populations may be observed that are infected ornot infected, that had a long-term exposure or chronic infection, thathad spontaneous recovery, that represents a group of responders (ornon-responders) to a particular drug treatment, or that had at leastpartial immunity to the pathogen.

In still further contemplated aspects, immunodominant antigens areidentified by selecting for an antigen (preferably within a well-definedsub-population) that (a) produces in at least 40-50% of a population ameasurable signal, and (b) has a signal strength of at least 40% of theoverall average signal intensity. However, and more preferably, thesignal strength will be at least above average of the overall averagesignal intensity, and even more preferably in the upper tertile(quartile, or even quintile) of signal intensities in the assay.Therefore, and viewed from another perspective, immunodominant antigenswill preferably be selected in a comparison of at least two series oftests, wherein one series of tests is typically the sub-population(e.g., primary infection, secondary infection, recovering, chronic,etc.) and the other series of tests is the control group (e.g., othersub-population or control group). Still further, it is generallypreferred that the series of tests also include a negative controlagainst which the potential immunodominant antigens are compared.

Consequently, and with particular respect to the pathogens presentedherein, it should be appreciated that compositions comprising one ormore selected immunodominant antigens can be prepared that will have astatistically high probability to elicit or have elicited an immuneresponse in a relatively large group of patients. Further, where theantigens are determined from selected sub-populations (e.g., recoveringpatients, chronic patients, primary infection, secondary infection,etc.), the antigens also have a known association with a diseaseparameter and thus allow staging of the disease and/or prediction oftherapeutic efficacy. Moreover, as the antigens presented herein areimmunodominant antigens, it should be noted that vaccine compositionscan be prepared with known or predictable immunogenicity.

For example, numerous antigens of Burkholderia pseudomallei (thoseencoded by nucleic acids SEQ ID NO:966 to SEQ ID NO:1150), Borreliaburgdorferi (those encoded by nucleic acids SEQ ID NO:546 to SEQ IDNO:637), Chlamydia muridarum (those encoded by nucleic acids SEQ ID NO:1to SEQ ID NO:134), Coxiella burnetii (those encoded by nucleic acids SEQID NO:678 to SEQ ID NO:713), Francisella tularensis (those encoded bynucleic acids SEQ ID NO:663 to SEQ ID NO:677), Herpes virus 1, Herpesvirus 2, and Vaccinia virus (those encoded by nucleic acids SEQ IDNO:638 to SEQ ID NO:654, SEQ ID NO:655 to SEQ ID NO:662, and SEQ IDNO:714 to SEQ ID NO:748, respectively), Mycobacterium tuberculosis(those encoded by nucleic acids SEQ ID NO:749 to SEQ ID NO:965),Plasmodium falciparum (those encoded by nucleic acids SEQ ID NO:135 toSEQ ID NO:445), and Brucella melitensis (those encoded by nucleic acidsSEQ ID NO:446 to SEQ ID NO:545) were identified as immunodominant (seealso examples and protocol below). With respect to the reading frame foreach of the sequences of SEQ ID NO:1 to SEQ ID NO:1150, it should benoted that the first base in the sequences is either the first base ofthe start codon or the first base in the first codon of the polypeptidethat was identified with the methods and compositions provided herein.Most typically, the last three bases denote the stop codon, or the lastbase of the last codon of the polypeptide that was identified with themethods and compositions provided herein.

In these examples, each of the antigens was characterized, inter alia,with regard to their individual and relative reactivities for each ofthe pathogens. Most typically, reactivity was measured as strength ofimmunogenicity (e.g., such that average binding affinity and/or averagequantity of the antibodies produced a predetermined signal intensity(e.g., in the upper half, upper tertile, or even upper quartile)).Furthermore, each of the identified antigens was also characterized byassociation with at least one parameter. In most cases, the diseaseparameter was acute infection with the pathogen, and in further cases,the disease parameter was also primary and/or secondary infection.Therefore, it should be especially appreciated that identification ofimmunodominant antigens will not only allow for identification ofstatistically meaningful antigens for diagnosis, vaccine development,and treatment, but also allow to develop a stage and disease specifictool to identify candidate molecules to fine-tune diagnosis and/ortreatment.

For example, suitable diagnostic devices especially include thosecomprising one or more of the immunodominant antigens, fragments, oranalogs thereof that are encoded by nucleic acids according to SEQ IDNO:1 to SEQ ID NO:1150. Depending on the particular device format, thedevice may have only a single immunodominant antigen, fragment, oranalog that may be used for detection of binding of antibodies fromblood or serum in an automated manner or by visual observation. Forexample, where a single immunodominant antigen is employed, suitabledevices may be in the format of a dipstick or competitive ELISA. On theother hand, where multiple immunodominant antigens are employed,suitable devices may be in the format of an array that can be read in anautomated device (e.g., via scanner) or visual manner (e.g., dye-formingcolorimetric reaction). Most typically, in such devices, the pluralityof antigens is deposited in a spatially addressable manner (e.g., x-ymatrix or mixed beads with color association). Moreover, it should benoted that diagnostic devices contemplated herein may be based onnumerous well known manners of detection, including ELISA (sandwich ornon-sandwich), competitive ELISA, anti-idiotypic antibodies, etc.,wherein all known colorimetric and photometric (e.g., fluorescence,luminescence, etc.) or radiometric reactions are deemed suitable foruse.

In most typical devices, a plurality of immunodominant antigens of asingle (or multiple) pathogen and/or serotype are deposited onto anaddressable solid phase and exposed to blood, serum, plasma or otherantibody-containing body fluid. Consequently, so prepared compositionscan be employed to identify and/or characterize an immune response of anindividual against selected antigens, and optionally assess the kind ofimmune response (e.g., identification of early, mid, late, or chronicinfection), as well as disease progression, efficacy of therapy, etc.Most typically, the plurality of antigens will include at least 2 to 10antigens, but significantly higher amounts of antigens are alsocontemplated, including at least 25%, more typically at least 50%, evenmore typically at least 75%, and most typically at least 90% of theproteome of the pathogen. In further typical aspects of the inventivesubject matter, contemplated arrays are most preferably processed in amicrofluidic device. For example, an array of antigens in such devicesmay be printed on a membrane or other material (e.g.,nitrocellulose-coated carrier of less than 1 cm² area) that is thenplaced in a microfluidic device having sample/reagent inlet and outletports. Depending on the specific configuration, signals may be acquiredusing optical methods (e.g. CCD chip, flat bed scanner, etc.),electrical methods (e.g., voltametric or amperometric), or other methodswell known in the art. Alternatively, visual detection or detectionusing a regular flat bed scanner at 1200 dpi resolution and/orfluorescence detection is also deemed suitable.

In another example, immunodominant antigens according to the inventivesubject matter may also be employed to generate an antibody preparationthat can be used as a passive vaccination for therapeutic treatment of adisease caused by the above pathogens.

In still further contemplated aspects, the immunodominant antigenspresented herein may also be employed in the manufacture of a vaccinethat comprises at least one, and more typically at least two of theimmunodominant antigens encoded by nucleic acids according to SEQ IDNO:1 to SEQ ID NO:1150. More preferably, however, contemplated vaccineswill include between two and five, or at least six, and even moreantigens, of which at least one of the antigens is an immunodominantantigen. Such vaccine compositions may be directed to elicit immunityagainst single or multiple serotypes and may thus comprise distinctimmunodominant antigens, optionally from multiple and distinct serotypesand/or species. Moreover, it should be appreciated that vaccines may beproduced that predominantly, or even exclusively, compriseimmunodominant antigens of a single parameter. For example, a vaccinemay comprise immunodominant antigens that are characteristic for apopulation that has recovered from an infection with the pathogenwithout drug intervention. In less preferred aspects, the sequencesaccording to SEQ ID NO:1 to SEQ ID NO:1150 may also be employed as DNAvaccines, or be part of an in vivo expression system that triggers animmune response against the in vivo produced recombinant antigen orfragment thereof.

With respect to suitable formulations of vaccines, it should berecognized that all known manners of producing such vaccines are deemedappropriate for use herein, and a person of ordinary skill in the artwill be readily able to produce such vaccines without undueexperimentation (see e.g., “Vaccine Adjuvants and Delivery Systems” byManmohan Singh; Wiley-Interscience (Jun. 29, 2007), ISBN: 0471739073; or“Vaccine Protocols” (Methods in Molecular Medicine) by Andrew Robinson,Martin P. Cranage, and Michael J. Hudson; Humana Press; 2 edition (Aug.27, 2003); ISBN: 1588291405). Therefore, suitable vaccines may beformulated as injectable solutions, or suspensions, intranasalformulations, or as oral formulations.

Examples

The following examples are provided for Plasmodium falciparum (Pf) and,in part, also for Vaccinia virus as target organism. Substantiallysimilar protocols were adapted to identify immunodominant antigens fromBurkholderia pseudomallei, Borrelia burgdorferi, Brucella melitensis,Chlamydia muridarum, Coxiella burnetii, Francisella tulare, Herpes virus1 and 2, and Mycobacterium tuberculosis.

Genes/ORFs for Pf were selected in silico and associated (where known)with various specific criteria, including pattern of stage-specific geneor protein expression deduced from genomic, proteomic, or cell biologydatasets. PCR primers were then computed to allow for amplification,cloning, and expression as further outlined below. So selectedgenes/ORFs were then amplified cloned using a high throughput PCRcloning method as previously described by the inventors (Genome Res 14,2076-2082 (2004)). Efficiencies of the overall process of PCRamplification and cloning were typically about 90% (with higher valuesfor smaller genes and lower values for larger [>2 kBp] genes). A subsetof the amplified and cloned genes were sequenced to confirm the identityof the target gene and to verify that mutations were not introduced as aresult of the amplification and cloning process. On average, 97%sequence identity was noted.

As compared with other organisms, Pf proteins have often been difficultto express by conventional methodologies in bacterial, yeast or insectexpression systems. The inventors therefore evaluated the efficiency ofPf protein expression in an E. coli based cell-free in vitrotranscription/translation system (RTS 100, commercially available fromRoche Biosciences) using 295 different cloned and HIS-tagged Pf ORFs astemplates. Quantification of the signal intensities revealed that 94% ofsignals were ≧4x the controls. A replicate HA-tagged Pf array wasproduced with two sets of Pf vectors, containing a stop codon eitherbefore or after the HA tag. Such tagged recombinant proteins allowfacile identification, rough quantification, and purification wheredesired. Uniformly high expression efficiencies >90% were obtained inall cases. Remarkably, the amount of protein printed per chip isessentially at saturation (data not presented), meaning that differencesbetween any detected antigen reactivities would not be due todifferences in the amount of protein spotted.

PCR amplified and cloned genes/ORFs were expressed in the RTS100cell-free in vitro transcription/translation system, and printed induplicate onto microarray chips. These chips were probed with sera froma population of subjects (n=12) who were naturally exposed to malaria ina hyperendemic region of Kenya, a population of subjects (n=10) who wereexperimentally immunized with radiation attenuated Pf sporozoites andeither protected (n=6) or not protected (n=4) against challenge withinfectious sporozoites, or a population of malaria-naïve individuals.

Most notably, sera from malaria exposed individuals and sporozoiteimmunized subjects reacted against different subsets of expressedproteins. For the naturally exposed group, there were 156 antigens withp values <1e-3 and 77 antigens with p values <1e-7 while for thepre-challenge sera from the irradiated sporozoite immunized groups,there were 17 antigens with p values <1e-3 in the protected group andnine antigens in the unprotected group. The low p values indicate thatthe signals obtained from the chip are highly significant. Backgroundreactivity from naïve donors was low (data not presented).

A similar panel was prepared for vaccinia virus with primary andsecondary exposure and with a variola as a control group. The results ofthis series of experiments can be taken from the summary panel of FIG.1, in which the different populations provided different targets fordifferent types of antibodies. Here, a proteome-wide view is providedfor the serological response to DryVax vaccinia and smallpox. Proteinmicroarrays displaying 210 different vaccinia strain WR proteins wereprobed with sera from individuals before, and 28-30 days after, primaryor secondary vaccination with DryVax. All signal intensities have beenbackground subtracted of control spots that lack template DNA, andassigned a color, with the dark end of the spectrum representing thehighest signals. At the low end of the scale, a signal intensity of 5000has been used as the cut-off above which a response was consideredpositive. Thus all negative responses are light. No significantreactivity was seen with any of the secondary antibodies alone (notshown). The combined IgG, IgM and IgA profiles in primary and secondaryDryVax infections include 64 and 49 different antigens (˜29% and ˜22% ofthe proteome), respectively. Interestingly, most antigens are recognizedby fewer than half the vaccinees, and late antigens dominate bothprimary and secondary responses. Despite variola being a differentorthopox species, antibody profiles of individuals infected withsmallpox are essentially indistinguishable from DryVax or VIG profiles.

As can be taken from FIG. 2, the antibody profiles between individualsresponding to the DryVax are heterogeneous to a significant degree. Eachprotein in the proteome was scored seropositive if its signal intensitywas >5000 after subtraction of its signal intensity seen with pre-immuneserum. As is readily apparent, there were relatively few antigens thatwere frequently recognized. Indeed, most positive antigens wererecognized by fewer than half the vaccinees, reflecting theheterogeneity of individual profiles, and with that reflecting thedifficulty in traditional vaccine development to obtain a vaccine thatis effective for a large fraction of a population. FIGS. 3A and 3Bdepict immunodominance profiles of vaccinia antigens in human primaryand secondary responses. Average signal intensities from sera 28-30 dayspost vaccination, with corresponding pre-vaccination signals, wereplotted as raw data. Primary responses are shown in panel (A) andsecondary responses are in panel (B). In each panel, a cut-off,indicated by the horizontal line, was set by the average of 7 pairs ofcontrol spots (expression reactions lacking DNA template) plus 2standard deviations. The relatively large standard deviations are due tothe inter-individual heterogeneity. Pre and post responses to an antigenthat are significantly different by 2-tailed, paired t-test areindicated as *** p<0.0005, ** p<0.005, * p<0.05. Others were considerednon-significant.

Characteristic profiles of antigen reactivity were noted for each of theclinically distinct cohorts, indicating that immunoreactive antigensidentified in this way may be useful for serodiagnostic tests that arenot only relevant with respect to the observed reactivity, but also withrespect to a disease parameter (e.g., previous exposure to the pathogen,duration of exposure to the pathogen, chronic infection, at leastpartial immunity to infection with the pathogen, expected positiveoutcome upon treatment, etc.). Remarkably, and with regard to the Pfimmune panel, the naturally exposed cohort who was protected againstclinical disease, but not Pf parasitemia, gave an entirely differentimmunoreactivity profile compared with the sporozoite-vaccinated cohortthat has both anti-disease and anti-parasite immunity. Further,naturally exposed subjects reacted more strongly to a larger number ofantigens than the sporozoite immunized subjects, and the subset ofsporozoite vaccinees that were protected reacted more strongly to alarger number of antigens than the unprotected group. Still further, theantigen repertoire in the sporozoite immunized and protected groupimmediately prior to challenge was unchanged after challenge. Incontrast, immunized volunteers who developed clinical malaria followingexperimental challenged (experimentally infected) developed anadditional subset of antibodies after challenge; many were similar tothe naturally exposed profile but some were unique to the experimentallyinfected group.

In addition to the subset of antigens identified on the basis ofintensity of response, a subset of antigens which were lessimmunoreactive but nonetheless frequently recognized was identified. Forexample, for naturally Pf exposed subjects, 121 antigens were recognizedat p<0.05 and frequency ≧50% regardless of signal intensity; 20 antigenswere recognized by 12/12 subjects (11/20 at signal intensities <4.0).For irradiated sporozoite immunized subjects 82 antigens were recognizedpre-challenge at p<0.05 and frequency ≧50% by both protected andunprotected subjects; five antigens were recognized by 10/10 subjects(2/5 at signal intensities <4.0). It is therefore contemplated thatantigens recognized in high frequency but not necessarily at highmagnitude may represent good diagnostic targets.

To illustrate how immunodominant antigen sets identified in this way canbe used for serodiagnostics, a protein microarray containingimmunodominant antigens from several infectious agents was fabricated.The antigens were expressed in the cell-free in vitrotranscription/translation system, printed onto FAST slides withoutfurther purification, and probed with anti-histidine primary antibodyfollowed by alkaline phosphatase conjugated secondary antibody. Theslide was developed with alkaline phosphatase substrate and scannedusing an ordinary desktop document scanner. The result of this scan isillustrated in FIG. 4A depicting the ‘antigen-loading’ of the array.Immunodominant antigens represented on this ‘diagnostic chip’ arederived from: F. tularensis (Ft), B. pseudomallei (Bp), B. burgdorferi(Bb), M. tuberculosis (TB), P. falciparum (If) and vaccinia virus (Vv).The negative control spots (within circles) correspond to in vitroexpression reactions without plasmid template. An example of the sameslide probed with serum from one of the Kenyan subjects naturallyexposed to Pf is shown in FIG. 4B. The strongest signals were againstthe Pf immunodominant antigen set, with weak reactivity toward vacciniavirus and M. tuberculosis antigens. Reactivity against non-Pf antigenscould be a consequence of prior exposure to these infectious agents orto cross-reactivity.

The following exemplary protocol is provided to illustrate the steps andreagents used in the identification of the immunodominant antigens of Pfpresented herein. Unless expressly stated, standard laboratorytechniques well known to a person of ordinary skill in the art wereemployed.

PCR Amplification of Linear Acceptor Vector

Plasmid pXT7 (10 μg; 3.2 kb, KanR) was linearized with BamHI (0.1 μg/μ1DNA/0.1 mg/ml BSA/0.2 units/μl BamHI; 37° C. for 4 hr; additional BamHIwas added to 0.4 units/μl at 37° C. overnight). The digest was purifiedusing a PCR purification kit (Qiagen, Valencia, Calif.), quantified byfluorometry using Picogreen (Molecular Probes, Carlsbad, Calif.)according to the manufacturer's instructions, and verified by agarosegel electrophoresis (1 μg). One ng of this material was used to generatethe linear acceptor vector in a 50-μl PCR using 0.5 μM each of primers5′-CTACCCATACGATGTTCCGGATTAC (SEQ ID NO:1151) and5′-CTCGAGCATATGCTTGTCGTCGTCG (SEQ ID NO:1152), and 0.02 units/μl Taq DNApolymerase (Fisher Scientific, buffer A)/0.1 mg/ml gelatin (Porcine,Bloom 300; Sigma, G-1890)/0.2 mM each dNTP with the followingconditions: initial denaturation of 95° C. for 5 min; 30 cycles of 95°C. for 0.5 min, 50° C. for 0.5 min, and 72° C. for 3.5 min; and a finalextension of 72° C. for 10 min.

PCR Amplification of ORF Insert

A total of 1-10 ng of Pf genomic DNA (3D7 strain) was used as templatein a 50-μl PCR. The following primers were used (0.5 μM each):5′-CATATCGACGACGACGACAAGCATATGCTCGAG (SEQ ID NO:1153; 20-mer ORFspecific at the 5′ end) and 5′-ATCTTAAGCGTAATCCGGAACATCGTATGGGTA (SEQ IDNO:1154; 20-mer ORF specific at the 3′ end). The Pf genome is the mostA+T rich genome sequenced to date with an overall (A+T) composition of80.6%, rising to ˜90% in introns and intergenic regions. Consequently,PCR amplification of Pf genes using genomic DNA template wasproblematic. Initially, PCR was carried out using regular Taq DNApolymerase: 0.02 units/μl TaqDNA polymerase (buffer A, FisherScientific)/0.1 mg/ml gelatin (Bloom 300, Porcine; G-1890, Sigma)/0.2 mMeach dNTP. Conditions were as follows: initial denaturation of 95° C.for 5 min; 30 cycles of 20 sec at 95° C., 30 sec at 50° C., and 60sec/kb at 72° C. (1-3 min on average, based on ORF size); and a finalextension of 72° C. for 10 min. PCR products that were more difficult toproduce were reamplified by using a 30 sec annealing time at 45° C. or40° C., instead of 30 sec at 50° C. Also, the extension temperature wasdecreased from 65-72° C. to 50° C. Subsequently PCR products wereobtained using a Taq polymerase with improved proof-readingcharacteristics (Triplemaster from Eppendorf), increasing the efficiencyof the PCR step to 87%: 0.04 units/μl Triple Master PCR system(high-fidelity buffer, Eppendorf)/0.4 mM each dNTP (Eppendorf).Conditions were as follows: initial denaturation of 95° C. for 3 min; 35cycles of 15 sec at 95° C., 30 sec at 40° C., and 60 sec/kb at 50° C.(1-3 min on average, based on ORF size); and a final extension of 50° C.for 10 min., PCR products that were difficult were reamplified using 50ng genomic DNA. The PCR product was visualized by agarose gelelectrophoresis (3 μl). For quantification, the product was purified(PCR purification kit, Qiagen) and quantified by fluorometry. Since thereliability of producing the desired PCR product decreases as the lengthof the genomic DNA fragment increases, exons longer than 3,000 bp weredivided into multiple overlapping sections, with 50 nucleotide overlaps.

In Vivo Recombination Cloning

Competent cells were prepared in our laboratory by growing DH5α cells at18° C. in 500 ml of SOB (super optimal broth) medium (2% tryptone/0.5%yeast extract/10 mM NaCl/2.5 mM KCl/20 mM MgSO₄) to an OD of 0.5-0.7.The cells were washed and suspended in 10 ml of pre-chilled PCKMS buffer(10 mM Pipes/15 mM CaCl₂/250 mM KCl/55 mM MnCl₂/5% sucrose, pH 6.7) onice, and 735 μl of DMSO was added dropwise with constant swirling. Thecompetent cells were frozen on dry ice—ethanol in 100-μl aliquots andstored at −80° C. Each transformation consisted of the following: 10 μlof competent DH5α and 10 μl of DNA mixture (40 ng of PCR-generatedlinear vector/10 ng of PCR-generated ORF fragment; molar ratio, 1:1;vector, 1-kb ORF fragment). For transformation, the purification of PCRproduct was unnecessary. The mixture was incubated on ice for 45 min,heat shocked at 42° C. for 1 min, and chilled on ice for 1 min; mixedwith 250 μl of SOC (super optimal catabolizer) medium (2% tryptone/0.55%yeast extract/10 mM NaCl/10 mM KCl/10 mM MgCl₂/10 mM MgSO₄/20 mMglucose); incubated at 37° C. for 1 hr; diluted into 3 ml of LB mediumsupplemented with 50 μg of kanamycin per ml (LB Kan 50); and incubatedwith shaking overnight. The plasmid was isolated and purified from thisculture, without colony selection.

In Vitro Protein Expression

Plasmid templates used for in vitro transcription/translation wereprepared by using QIAprep Spin Miniprep kits (Qiagen), including the“optional” step, which contains protein denaturants to deplete RNaseactivity. In vitro transcription/translation reactions (RTS 100Escherichia coli HY kits; Roche) were set up in 25 μl PCR 12-well striptubes and incubated for 5 h at 30° C., according to the manufacturer'sinstructions.

Immuno-Dot Blots

To assess relative efficiency of protein expression, 0.3 μl of wholerapid-translation system (RTS) reactions were spotted manually ontonitrocellulose and allowed to air dry before blocking in 5% nonfat milkpowder in TBS containing 0.05% Tween 20. Blots were probed withhyperimmune sera diluted to 1:1,000 in blocking buffer with or without10% E. coli lysate. Routinely, dot blots were stained with both mouseanti-poly-HIS mAb (clone, HIS-1; H-1029, Sigma) and ratanti-hemagglutinin (HA) mAb (clone, 3F10; 1 867 423, Roche), followed byalkaline phosphatase-conjugated goat anti-mouse IgG (H+L) (BioRad) orgoat anti-rat IgG (H+L) (Jackson ImmunoResearch) secondary Abs,respectively. Bound human Abs were visualized with nitroblue tetrazolium(nitro-BT) developer to confirm the presence of recombinant protein.

Microarray Chip Printing

For microarrays, 10 μl of 0.125% Tween 20 was mixed with 15 μl of RTSreaction (to a final concentration of 0.05% Tween 20), and 15-μl volumeswere transferred to 384-well plates. The plates were centrifuged at1,600×g to pellet any precipitate, and supernatant was printed withoutfurther purification onto nitrocellulose-coated FAST glass slides(Schleicher & Schuell) by using an OmniGrid 100 microarray printer(Genomic Solutions, Ann Arbor, Mich.). All ORFs were spotted induplicate to enable statistical analysis of the data. Data valuesreported herein represent the average of pairs. In addition, each chipcontained an area printed with controls consisting of RTS reaction usingno DNA.

Protein Microarray Screening

Microarray chips were probed with human serum that was firstpre-absorbed against E. coli-lysate to block anti-E. coli antibodies asdescribed previously. In the absence of pre-absorbing, high titers ofanti-E. coli antibodies could mask any antigen-specific responses whenusing whole RTS reactions on dot blots and arrays. For all staining,slides were first blocked for 30 min in protein array-blocking buffer(Schleicher & Schuell) and then incubated in serum for 2 hr, at roomtemperature. Antibodies were visualized with Cy3-conjugated secondaryAbs (biotinylated secondary followed by Streptavidin PBXL-3, forHIS-probing) (Jackson ImmunoResearch) and scanned in a ScanArray 4000laser confocal scanner (GSI Lumonics, Billerica, Mass.). Fluorescenceintensities were quantified by using QuantArray software (GSI Lumonics).Other studies in our affiliated University laboratory have establishedthat the results from scanned microarray chips can be representednumerically and that signal intensity is proportional to antibody titer(data not shown).

ELISA

To validate the immunoreactivity detected by the protein microarrays,sera was analyzed by ELISA against a panel of known andwell-characterized Pfpre-erythrocytic stage antigens (PfCSP, PfLSA1, andPfExp1) and erythrocytic stage antigens (PfAMA1, PfMSP1), as previouslydescribed. The mean OD readings of quadruplicate assays were recorded,and results reported as the OD value at each serum dilution and asendpoint dilution (defined as greater than the mean+/−3 standarddeviation of negative control sera).

Indirect Fluorescent Antibody Test (IFAT)

Antibody recognition of Pf (NF54/3D7) sporozoite or blood stageparasites was evaluated by IFAT as described previously. Reactivity wasscored as positive when the immunofluorescence pattern of the parasitewas recognized and when the fluorescence was above the background of thenegative controls. IFAT results were expressed as the endpoint serumdilution at which positive fluorescence was detected.

Malaria-Exposed Study Populations

Individuals were selected for study on the basis of malaria history.Studies were conducted in compliance with all applicable Federalregulations governing the protection of human subjects. The irradiatedsporozoite study protocol was approved by the Naval Medical ResearchCenter Committee for the Protection of Human Subjects, the Office of theSpecial Assistant for Human Subject Protections at the Naval Bureau ofMedicine and Surgery, and the Human Subjects Research Review Board ofthe Army Surgeon General. The Kenyan samples were collected under astudy protocol approved by the Naval Medical Research Institute'sCommittee for the Protection of Human Subjects, the Walter Reed ArmyInstitute of Research Human Use Committee, and the Kenya MedicalResearch Institute/National Ethical Review Committee. Written informedconsent was obtained from all subjects.

Sporozoite Immunized Volunteers

Caucasian volunteers (n=10) were experimentally immunized withradiation-attenuated Pf sporozoites as previously described (The Journalof Infectious Diseases (2002), Vol: 185, p1155-64). Subjects werechallenged by the bites of 5 infected Anopheline mosquitoes, andevaluated for the development of clinical malaria. Protection wasdefined as complete absence of blood-stage parasitemia (sterileprotection). Six of the 10 immunized volunteers were protected againstsporozoite challenge and were classified as sporozoite-immune; four werenot protected and were classified as sporozoite-exposed but non-immune.Serum samples were collected from each volunteer prior to immunization(pre-bleed), at the completion of the immunization series andimmediately prior to challenge (pre-challenge), and following challenge(post-challenge). Pre- and post-challenge IFAT titers against Pfsporozoites were 613.3 (mean; range 160-1280) and 170 (mean; range40-320) for protected and unprotected volunteers, respectively.

Individuals Naturally Exposed to Malaria

Kenyan subjects (n=12) were residents of the Asembo Bay area of Kenya.In this area, the year round prevalence of Pf infection amongst children6 months to 6 years of age has been documented as 94.4-97.8%. Enrolledsubjects reported an average of 2.1 episodes of clinical malaria withinthe previous year. The study cohort derives from a subset of 185volunteers previously enrolled in an immunoepidemiology study andselected for the current study on the basis of sex, age, malaria historyand recognition of native Pf sporozoites and parasitized erythrocytes,by IFAT. Pf sporozoite and blood-stage IFAT titers for the pool ofhyperimmune sera from these 185 individuals were 5,120 and 81,920,respectively.

Analysis of Individual Array Measurements

As a first step in determining significantly bound antigens for eachserum/array using statistical tests, the inventors defined the truenegative control signal to compare each antigen with the mean signal ofall spotted controls on the array. Since comparisons needed to becarried across arrays, the inventors transformed the raw signals usingthe vsn (asinh transformation, similar to log for higher intensities)method, shown to effectively calibrate array measurements throughshifting and scaling and also to stabilize the variance in DNAmicroarray and 2D difference gel electrophoresis data analyses. Becausestandard deviation (SD) estimates can be unreliable (artificially highor low) when there is low replication of measurements, and since eachantigen was spotted only twice per array, the inventors applied theBayes-regularization technique described in Baldi and Long(Bioinformatics (2001), 17, 509-519; Baldi, P. and Hatfield, G. W.(2002) DNA Microarrays and Gene Expression: From Experiments to DataAnalysis and Modeling, Cambridge University Press, Cambridge, UK; andBioinformatics (2006), Vol. 22, No. 14; p:1760-1766, all incorporated byreference herein). This technique derives more robust estimates (asshown in the context of DNA microarray data analysis) of the SD of eachantigen as a weighted combination of the sample SD and the pooled SD ofneighboring antigens with similar signal intensity. Using these newregularized estimates for the standard deviation, we conducted a seriesof Bayes-regularized one-sided t-tests on antigens with higher meansignal than the defined control to reliably estimate the signal changesbetween each antigen and control, and computed the correspondingp-values.

Analysis of Groups of Array Measurements

In addition to determining the positive antigens recognized by each ofthe individual sera, the inventors averaged replicated spot measurementsper sera and pooled the responses for each cohort/group, to identify thepositive antigens while taking into account the biological variationwithin the sera in each group. Since the measurements were obtained fromdifferent arrays, the inventors performed a calibration and variancestabilization of the measurements using the vsn method (Bioinformatics18 Suppl 1, S96-104 (2002) prior to the pooling of measurements. As forthe analysis of individual sera, the inventors defined the true negativecontrol using the mean control signal spotted on the arrays. One-sidedBayes-regularized t-tests were performed within each group to compareand rank the antigens with a higher mean signal than the control. Forthe individual and cohort/group analysis, using the average SD of 5-30neighboring antigens along with a weight of 5 “pseudocounts” forcomputing the Bayes-regularized SD was observed to achieve a moderateregularization effect. Given the large number of hypotheses beingtested, the inventors applied the method in Storey and Allison et. al.to the set ofp-values to estimate the experiment-wide false discoveryrates (FDR). For the individual and cohort analyses of the 43 arrays, ap-value cutoff of 0.05 corresponded to an FDR level of 0.06-0.065. Withthe additional criteria applied for determining a positive response asdescribed below, the inventors expect the FDR to be lower.

Analysis of Frequency of Response

In addition to analyzing the intensity of response (as described above),the inventors also assessed the frequency of response for each antigenas the number of individuals within a given cohort for which thatantigen was positive on the basis of normalized signal intensityrelative to control.

Criteria of Positivity (Immunodominance)

Final classification of antigen reactivity was made taking into accountboth magnitude of response (signal intensity) and frequency ofrecognition. The responses by a particular cohort of donors wereconsidered positive overall if all of the following criteria were met:(1) normalized signal intensity >4.0 (ratio of signal intensity of testrelative to control >4.0); (2) response was statistically significant(p<0.05) compared with control signal intensity; and (3) frequency ofpositive responses within a particular cohort ≧2.0.

Thus, specific embodiments and applications of immunodominantcompositions and methods have been disclosed. It should be apparent,however, to those skilled in the art that many more modificationsbesides those already described are possible without departing from theinventive concepts herein. The inventive subject matter, therefore, isnot to be restricted except in the spirit of the appended claims.Moreover, in interpreting both the specification and the claims, allterms should be interpreted in the broadest possible manner consistentwith the context. In particular, the terms “comprises” and “comprising”should be interpreted as referring to elements, components, or steps ina non-exclusive manner, indicating that the referenced elements,components, or steps may be present, or utilized, or combined with otherelements, components, or steps that are not expressly referenced.Furthermore, where a definition or use of a term in a reference, whichis incorporated by reference herein is inconsistent or contrary to thedefinition of that term provided herein, the definition of that termprovided herein applies and the definition of that term in the referencedoes not apply.

SEQUENCE LISTING

The Sequence Listing providing sequences with the SEQ ID NO:1 to SEQ IDNO:1150 is submitted as a single file on a single compact disc incomputer readable format (CRF; three copies of the disc were submittedtogether with the original CRF disc), wherein the single file isentitled “101519.0001PCT Sequence Listing ST25.txt”, which was createdOct. 25, 2007, which has a size of 1908 kb, and which is incorporated byreference herein. Therefore, four identical discs, each containing asingle sequence listing file with the file name “101519.0001PCT SequenceListing ST25.txt” have been submitted.

What is claimed is:
 1. An isolated antigen composition comprising: aplurality of immunodominant antigens of a pathogenic organism associatedwith a carrier; wherein the antigens have quantified and known relativereactivities with respect to sera of a population infected with theorganism; wherein the antigens are determined to have a knownassociation with a disease parameter; and wherein the plurality ofantigens are encoded by nucleic acids selected from the group consistingof SEQ ID NO:633 (BB_A19), SEQ ID NO:575 (BB_A25), SEQ ID NO:595(BB_K07), SEQ ID NO:596 (BB_K12), SEQ ID NO:14 (BB_(—)0147), SEQ IDNO:550 (BB_(—)0279), and SEQ ID NO: 637 (VIsE), or fragments thereof. 2.The antigen composition of claim 1 further comprising at least oneadditional antigen of Borrelia burgdorferi, encoded by nucleic acidsselected from the group consisting of SEQ ID NO:546 to SEQ ID NO:637. 3.The antigen composition of claim 1 wherein the carrier is apharmaceutically acceptable carrier, and wherein the composition isformulated as a vaccine.
 4. The antigen composition of claim 3 whereinthe vaccine comprises at least four antigens.
 5. The antigen compositionof claim 3 wherein the vaccine comprises antigens from at least twodistinct pathogens.
 6. The antigen composition of claim 3 wherein theantigens or fragments thereof are recombinant.
 7. The antigencomposition of claim 3 wherein the antigens or fragments thereof are atleast partially purified.
 8. The antigen composition of claim 1 whereinthe carrier is a solid carrier, and wherein the plurality of antigens isdisposed on the carrier in an array.
 9. The antigen composition of claim8 wherein the antigens are from at least two distinct pathogens.
 10. Theantigen composition of claim 8 wherein the antigens or fragments thereofare at least partially purified.
 11. A method for the detecting presenceof antibodies which specifically bind to antigens of Borreliaburgdorferi, and which are present in a bodily fluid sample, comprisingcontacting the sample with antigens of Borrelia burgdorferi, wherein theantigens are encoded by at least two of nucleic acids SEQ ID NO:633(BB_A19), SEQ ID NO:575 (BB_A25), SEQ ID NO:595 (BB_K07), SEQ ID NO:596(BB_K12), SEQ ID NO:14 (BB_(—)0147), SEQ ID NO:550 (BB_(—)0279), and SEQID NO: 637 (VIsE), and detecting antibodies which bind to the antigens.12. The method of claim 11, wherein the antigens are present in a crudeexpression extract or in partially purified form.
 13. The method ofclaim 11, wherein the step of detecting the antibodies comprises use ofa signal-generating anti-antibody.
 14. The method of claim 11, whereinbinding affinity of respective antibodies which specifically bind toantigens of Borrelia burgdorferi, are known and indicative of anactivity state of lyme disease.
 15. The method of claim 11, wherein theantigens of Borrelia burgdorferi, are coupled to a solid phase prior tothe step of contacting the sample with the antigens.
 16. The method ofclaim 15, wherein the antigens of Borrelia burgdorferi, are coupled tothe solid phase in an array.
 17. The method of claim 11, wherein atleast one of the antigens encoded by at least two of nucleic acids SEQID NO:633 (BB_A19), SEQ ID NO:575 (BB_A25), SEQ ID NO:595 (BB_K07), SEQID NO:596 (BB_K12), SEQ ID NO:14 (BB_(—)0147), SEQ ID NO:550(BB_(—)0279), and SEQ ID NO: 637 (VIsE) is present as anantibody-binding fragment.
 18. The method of claim 11, wherein theantigens further comprise at least one antigen of Borrelia burgdorferi,that is encoded by a sequence selected from the group consisting of SEQID NO:546 to SEQ ID NO:637, or antibody binding fragments thereof.
 19. Adiagnostic device for detection presence of antibodies whichspecifically bind to antigens of Borrelia burgdorferi and which arepresent in a bodily fluid sample, comprising a solid phase to whichantigens are coupled, wherein the antigens are encoded by at least twoof nucleic acids selected from the group consisting of SEQ ID NO:633(BB_A19), SEQ ID NO:575 (BB_A25), SEQ ID NO:595 (BB_K07), SEQ ID NO:596(BB_K12), SEQ ID NO:14 (BB_(—)0147), SEQ ID NO:550 (BB_(—)0279), and SEQID NO: 637 (VIsE), and wherein the device is suitable for detection ofthe presence of the antibodies according to claim
 11. 20. The diagnosticdevice of claim 19, wherein binding of the antibodies is indicative ofactive lyme disease.